1. Field of the Invention
The present invention relates to prothrombin time reagents for monitoring extrinsic blood coagulation activities, a method for preparing the reagent and a method for using the reagent. More particularly, the invention relates to a dried synthetic prothrombin time reagent prepared from recombinant tissue factor and synthetic phospholipids, and dried under ambient conditions.
2. Description of Related Art
Blood coagulation tests may be performed for a variety of purposes, including determination of the bleeding susceptibility of patients undergoing surgery and monitoring of patients undergoing anti-coagulation therapy for the prevention of blood clots. A number of coagulation tests presently are in use. One of these tests is the "prothrombin time" (PT) test. The PT test relies upon the induction of the extrinsic coagulation protease factor VIIa by thromboplastin in a blood sample to be tested. The extrinsic coagulation pathway results in the production of thrombin, a proteolytic enzyme that catalyzes the conversion of fibrinogen to fibrin, which is essential to the clotting process.
Thromboplastin, also known as tissue factor, is a membrane associated glycoprotein that forms a complex with blood coagulation factors VIIa. The complex formed enhances the proteolytic activity of these two factors. The functional activity of prothrombin depends on the presence of phospholipids (Bach, Ronald R., Initiation of Coagulation by Tissue Factor, CRC Critical Reviews in Biochemistry 1988; 23(4): pp.339-368). Once formed, the factor VIIa/tissue factor complex activates a series of specific enzymes that comprise the extrinsic and common pathways of the coagulation cascade, which ultimately lead to the formation of thrombin, fibrin, platelet activation, and finally clot formation (Nemerson, Yale, Tissue Factor and Hemostasis, Blood 1988; 71: pp. 1-8).
The prothrombin time (PT) test utilizes this series of enzymatic events in vitro under controlled conditions to diagnose dysfunctions or deficiencies in the blood coagulation system of patients. Other uses include the monitoring of patients undergoing anticoagulant therapy. The time period it takes for clot formation to occur is the Prothrombin Time, or PT value.
PT reagents are used to monitor the coagulation activities of the extrinsic pathway of plasma, including those from patients on Coumadin therapy. For the PT test, a highly sensitive reagent with an International Sensitivity Index (ISI) of 1.0 is desired. With an ISI of 1.0, the calculation of the International Normalized Ratio (INR) of PT coagulation test is simplified.
A PT reagent must have the following characteristics: sensitivity to abnormal samples, a well-defined normal PT value for normal plasma, providing accurate and reproducible results, maintaining consistency from lot to lot, and stability for storage in a dried state and upon reconstitution.
Tissues of vertebrates that have been added to citrated plasma and then recalcified accelerate clotting time. The tissue constituent that activates the coagulation protease cascade is commonly referred to as thromboplastin, or tissue factor (TF). Tissue factors employed in the present PT tests contain crude tissue factor extracted from natural sources. Natural sources include rabbit brain, rabbit brain/lung tissue mixtures, human placenta, or ox brain. Each of these sources has problems associated with them. For example, rabbit brain thromboplastin shows some seasonal variability, it varies from lot to lot, and is in relatively short supply. Human tissue factor may contain HIV or other viral diseases. Ox brain provides values that are much longer than those observed when employing tissue factor from alternative sources. The longer values may reflect differences in the ability of ox tissue factor to bind human factor VII. Crude tissue factor preparations from natural sources also contain other coagulation factors as contaminants. The contamination with coagulation factors results in coagulation factor assay curves that are less sensitive to factor-deficient plasmas.
Tissue factor requires phospholipids for functional activity. Phospholipids found in PT reagents generally are those that adhere to tissue factor when it is extracted from animal sources. For example, the extraction of rabbit brain results in the concurrent isolation of both tissue factor and naturally occurring phospholipids which are bound to the tissue factor in vivo and survive the extraction process. No additional lipids arc usually added to such extracted tissue factor. As a result, the nature, quantity, and quality of the lipids employed in the PT reagent will therefore vary depending upon the starting tissues and the extraction process, and will lead to lot to lot inconsistencies. The DADE.RTM. thromboplastin reagents, Thromboplastin C, C+, and IS, available from Dade Intemational, Inc. of Deerfield, Ill., are all based on extracts of acetone-dehydrated rabbit brain. Partially purified extracts are blended with specific mixtures of buffers and stabilizers. Since the partially purified tissue factor extract is not completely delipidated, the addition of lipids back into the extract is unnecessary. The nature and composition of the resulting lipids is not well defined and can vary from lot to lot.
Different thromboplastin preparations either improve or reduce discrimination between blood samples having different prothrombin times. Thromboplastins with greater discrimination are termed "more sensitive." The liquid phase sensitivity of a preparation is graded by use of the international sensitivity index. The value is found by plotting on a logarithmic scale, the prothrombin time seen with a thromboplastin lot in question versus the prothrombin time values seen with a standardized lot of thromboplastin. The ISI value is the slope of the resulting line multiplied by the ISI of the reference thromboplastin. More sensitive thromboplastins have lower ISI numbers around 1.0, and less sensitive thromboplastins have higher ISI numbers, typically around 2 to 3.
In attempting to avoid the problems associated with tissue factor from natural sources, the use of recombinant tissue factor for use in a PT reagent is described in U.S. Pat. No. 5,625,036 titled "Preparation of Prothrombin Time Reagents from Recombinant Human Tissue Factor and purified Natural and Synthetic Phospholipids" issued Apr. 29, 1997 to P. L. Hawkins, et al., incorprated herein by reference. As described in the patent, human tissue factor is cloned and expressed in a number of organisms including E. coli. A portion of the cloned tissue factor is employed in the PT reagent without loss of functional activity, since most of the intracellular (cytoplasmic) domain of the cloned tissue factor can be truncated. The PT reagent includes recombinant tissue factor, phospholipids, either synthetic or natural, calcium ion, and a buffer composition. Well known cryopreservatives may also be added such as trehalose, maltose, and mannitol. Hawkins and other prior art preparation methods invariably require lyophilization or freeze-drying of the reagent for stability upon storage.
U.S. Pat. No. 5,314,695 titled "Tissue Factor Based Prothrombin Time Reagent" issued May 24, 1994 to S. M. Brown, incorporated herein by reference, relates to a tissue factor prothrombin time reagent in which the tissue factor is inserted into the phospholipid bilayer of liposomes or phospholipid vesicles. A buffer containing a cryopreservative and glycine preferably forms part of the formulation. Either natural tissue factor or recombinant tissue factor can be used. The formulation composition is adjusted to allow maximum coagulant activity and sensitivity to extrinsic coagulation factors. Brown's described preparation methods also require lyophilization.
U.S. Pat. No. 5,358,853 titled "Liquid Thromboplastin Reagent" issued Oct. 25, 1994 to J. R. Butler, et al., included herein by reference, describes a stable liquid thromboplastin reagent with a long shelf life which is prepared without lyophilization. However, the crude rabbit brain extract and unpurified phospholipids which comprise the liquid thromboplastin reagent of U.S. '853 can affect sensitivity variability of the reagent with regard to the PT test.
U.S. Pat. No. 5,418,141 titled "Test Articles for Performing Dry Reagent Prothrombin Time Assays" issued May 23, 1995 to S. E. Zweig, et al., incorporated herein by reference, relates to test articles for performing dry reagent prothrombin time assays. The test articles comprise a solid phase membrane having dry, lyophilized thromboplastin immobilized therein. Coagulation neutral agents that facilitate rehydration of the dry thromboplastin are also provided on the solid phase membrane.
There remains a need therefore for a highly sensitive and consistent PT reagent that also remains stable upon drying without lyophilization or impregnation into a membrane matrix, thereby rendering it easier to manufacture, handle, transport, and store.